Phylogeography of the Patagonian otter Lontraprovocax: adaptive divergence to marine habitator signature of southern glacial refugia?Juliana A Vianna1,2,3*, Gonzalo Medina-Vogel1, Claudio Chehébar4, Walter Sielfeld5, Carlos Olavarría6,Sylvain Faugeron2
Abstract
Background: A number of studies have described the extension of ice cover in western Patagonia during the LastGlacial Maximum, providing evidence of a complete cover of terrestrial habitat from 41°S to 56°S and two mainrefugia, one in south-eastern Tierra del Fuego and the other north of the Chiloé Island. However, recent evidenceof high genetic diversity in Patagonian river species suggests the existence of aquatic refugia in this region. Here,we further test this hypothesis based on phylogeographic inferences from a semi-aquatic species that is a toppredator of river and marine fauna, the huillín or Southern river otter (Lontra provocax).
Results: We examined mtDNA sequences of the control region, ND5 and Cytochrome-b (2151 bp in total) in 75samples of L. provocax from 21 locations in river and marine habitats. Phylogenetic analysis illustrates two maindivergent clades for L. provocax in continental freshwater habitat. A highly diverse clade was represented byhaplotypes from the marine habitat of the Southern Fjords and Channels (SFC) region (43°38’ to 53°08’S), whereasonly one of these haplotypes was paraphyletic and associated with northern river haplotypes.
Conclusions: Our data support the hypothesis of the persistence of L. provocax in western Patagonia, south of theice sheet limit, during last glacial maximum (41°S latitude). This limit also corresponds to a strong environmentalchange, which might have spurred L. provocax differentiation between the two environments.
BackgroundClimate change caused substantial alterations of thelandscape and sea level, influencing patterns of speciesdistribution. Pleistocene glaciations, ice-sheet advancesand retreats in western Patagonia shaped land fragmen-tation, and the formation of islands and fjords along thePacific Coast [1]. Throughout the Last Glacial Maxi-mum (LGM,~25 000-15 000 years ago), ice sheetsextended from 56°S up to 35°S along the Andes moun-tain range, and to 41°S in lowland areas and at sea-levelin South America [2,3]. The southern fjords and chan-nels (SFC) of southern Chile (41°S to 56°S, Figure 1)were covered by an extensive ice sheet. The pollenrecord indicates major shifts in most species of plants inthis region [4]. As a result of the expected pattern of
species range contractions and subsequent expansionafter postglacial ice sheet retreat, genetic signatures oflow diversity and demographic expansions are expectedin these newly colonized areas [5,6]. This is the case fora vast majority of sigmodontine rodents, either fromlowlands, Patagonian mountains or Tierra de Fuego[7-9]. Other studies, however, have suggested the persis-tence of freshwater species in the region throughout thelast glacial period [10-13]. In the case of the Patagonianfreshwater fish Galaxias platei, distributed along thewestern side of the Andes (39°S - 49°S), survival couldhave occurred in a southern refuge, possibly due to dis-continuities of the ice field [12]. Likewise, southern refu-gia of the freshwater crab Aegla alacalufi has beensuggested at the El Amarillo hot springs, which was pos-sibly left uncovered by glacial ice, but isolated frommore northern refugia [13]. Thus, it seems populationsof freshwater species were not completely extirpated byice cover in southern areas. This alternative pattern
* Correspondence: [email protected] de Ecología y Biodiversidad, Facultad de Ecología y RecursosNaturales, Universidad Andrés Bello, Republica 440, Santiago, ChileFull list of author information is available at the end of the article
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addresses important questions about the history of thisregion, the magnitude of the ice cover extension, andthe consequences for the phylogeographic pattern ofspecies associated with the SFC system for which thereis a complete absence of data.In Chile, the northern glacial limit is also a boundary
for major environmental changes (e.g. topography,
currents, water salinity), resulting in a major biogeo-graphic transition for marine [14] and freshwater species[15]. This biogeographic boundary is marked by theoceanic divergence between the Humboldt and the CapeHorn current systems, contributing to the relative isola-tion of the SFC marine fauna [16,17]. The SFC ecosys-tem is supplied by marine currents such as those
Taitao Peninsula
Figure 1 Map of Lontra provocax distribution. Lontra provocax distribution in dark gray (Medina, 1996) and ice sheet coverage limit duringthe Last Glacial Maximum in dashed line (McCulloch et al., 2000), including the three southernmost sampling sites (19, 20, 21 see Table 1). Thehaplotypes are represented by different colours such as the figure 4.
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from subantarctic waters. These become mixed withfreshwater from abundant precipitation, river flow, andglacial meltwater [18]. Across the biogeographic bound-ary, hydrographic conditions change in freshwater envir-onments, based on the weather, slope and lithology [19].Two provinces are described for the biogeography of thefreshwater fishes: the Chilean province, with a southernarea of endemism from Valdivia River to Chiloé Island,and the Patagonian province, which is restricted to thewestern watersheds of continental Chiloé south toTierra del Fuego [15]. Different scenarios altered thespecies assemblages across biogeographical limits andlikely generated distinct patterns of genetic diversity.Phylogeographic inferences should help to understandspecies evolutionary history across marine and fresh-water biogeographic breaks. Here we use molecularmarkers to address the hypothesis of glacial refugia onan aquatic top predator, the Huillín or Southern riverotter, Lontra provocax. L. provocax populations extendacross the biogeographic boundary, from exclusivelyfreshwater habitat north of this limit to mostly marinehabitat in the SFC (Figure 1, 2).L. provocax has the smallest geographical range of all
otter species [20], being distributed across the Andean-Patagonian region of southern Chile and part of Argen-tina [21-23]. In continental waters, the species is foundfrom the Toltén River basin (39°S latitude) to the southof Chile [21]. The range extends through a limited areaof the Andes mountains into Nahuel Huapi NationalPark and Limay River in Argentina [24-26]. South ofChiloé Island in Chile (42°S) the species also occurs inmarine habitats along the SFC south to 56°S [27]. In theSFC south of Taitao Peninsula (46°S latitude), however,its distribution becomes exclusively marine [28,27]. AsL. provocax is highly dependent on the availability ofcrustacean prey [29,30], the absence of the species fromcontinental waters is related to the absence of crusta-ceans in the oligotrophic waters. L. provocax is solitarywith intrasexual territoriality and an average homerange of 11.3 km in rivers [31]. Dispersal is limited, asshown by radio-tracked otters, where the only long-range movement was made by a juvenile male thatmigrated 46 km downstream after release [31]. In fresh-water, occurrence of L. provocax is dependent on crusta-cean distribution, which is strongly influenced by theriver slope and altitude [32]. Consequently, the distribu-tion of L. provocax along rivers is mostly concentratedbelow 300 m altitude [33], which may limit gene flowacross the Andes mountain range. As crustacean andfreshwater fishes were able to live in southern Chileduring the LGM, L. provocax could have had a sufficientamount of food to survive during this period. However,the occurrence of L. provocax is also dependent onriparian vegetation. Terrestrial plants such as Fitzroya
cupressoides and Hypochaeris palustris were mostlyabsent along ice sheet cover areas during LGM [34,35].Thus, the question of L. provocax persistence duringLGM on southern Chile likely implies a trade offbetween availability of food and terrestrial habitat.So far the species has been studied mainly in the riv-
ers and lakes at the northern limit of its distribution.Comparisons between specimens living in freshwaterand marine environment are restricted to diet, and con-sists mainly of crustaceans such as Samastacus sp. andAegla sp. in rivers and lakes [24,29], shifting to marinefishes of the genus Patagonotothen sp., crustaceans [36]and sea urchins in the marine environment. Althoughotter species occur mostly in freshwater habitats, themajority of these species have also been recorded incoastal environments [30]. Otters distributed along thecoast, however, also need access to fresh water fordrinking and washing their dense fur to remove accu-mulating salt and maintain thermo-insulation [30].Fresh water is abundant along the SFC and L. provocaxhas also been recorded in inland rivers, such as QueulatRiver. It is important to note that, among all otter spe-cies, only two are exclusively adapted to marine habitats,i.e. the north Pacific Ocean sea otter (Enhydra lutris)and the chungungo or south Pacific marine otter(Lontra felina). Lontra felina recently diverged fromL. provocax, possibly from populations in the SFC thatprogressively adapted to the coastal marine habitat [37].The present study analysed the phylogeographic pattern
and the population structure of L. provocax, based on themitochondrial DNA sequences from control region (CR),the NADH dehydrogenase subunit 5 (ND5) and the cyto-chrome b gene (Cyt-b). We aimed to infer: i) the demo-graphic processes associated with the LGM south of thePleistocene ice cover limit for L. provocax populations;ii) the evolutionary relationship between freshwater andmarine populations; iii) the population structure withineach habitat type, among different continental riverbasins and across the Andes mountain range, comparingL. provocax populations from Chile and Argentina.
ResultsA total of 569 bp from the mitochondrial DNA controlregion (CR), 575 bp for ND5 and 1,007 bp of the cyto-chrome b (Cyt-b) were sequenced from a total of 75samples. A total of 11 CR haplotypes, 6 ND5 haplotypesand 9 Cyt-b haplotypes were found for Lontra provocax(Table 1). Although one CR haplotype did not illustratea clear geographic separation, ND5 and Cyt-b haplo-types showed a clear partition according to differentenvironments. Three ND5 haplotypes (I, II, III) wereexclusively from continental freshwater (CRL), whereashaplotype IV was distributed from the Chiloé Island riv-ers (CI) to the southern fjords and channels (SFC) and
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Nahuel Huapi area in Argentina. Haplotypes V and VIwere exclusively found in SFC (Table 1). On the otherhand, the Cyt-b haplotypes (A, B and I) found in bothcontinental and Chiloé Island freshwater habitats (CRLand CI), were not shared with SFC marine habitat,which showed a high diversity of Cyt-b haplotypes (C,
D, E, F, G, H; Table 1). The Partition Homogeneity Test(P = 0.1767) indicated statistical congruence betweenthe three mtDNA sequences (CR+ND5+Cyt-b), there-fore concatenated sequences were used for data analysis.Seventeen haplotypes and 26 polymorphic sites wereobserved from the concatenated mtDNA sequences.
Figure 2 Map of Lontra provocax samples collected in the northern part of the study area (1 to 18 according to Table 1). Ice sheetcoverage limit during the Last Glacial Maximum in dashed line (McCulloch et al., 2000). The haplotypes are represented by different colourssuch as the figure 4. Petrohue River is respresented by the location 9.
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The concatenated haplotype A-I-A (for CR-ND5-Cyt-b respectively) was widely distributed in the continentalfreshwater habitat, except in Petrohue where haplotypeC-III-A was observed. Haplotypes A-I-B and B-II-Awere also found only at Mahuidanchi River and QueuleRiver respectively. A unique haplotype A-IV-I was foundon Nahuel Huapi area in Argentina, not detected onChilean rivers. All samples from rivers on Chiloé Islandhad a unique haplotype A-IV-A, which was not found incontinental or marine sites. In the SFC, each haplotypewas restricted to one or few locations.The SAMOVA revealed increasing FCT values when
the numbers of groups increased (K = 2-6; FCT = 0.55-0.72). Petrohue River was separated out in all SAMOVApartitions. The most geographically coherent partitioncould be defined in 4 groups: (i) most continental riversand lakes locations including Chiloé Island (location1 to 11), (ii) continental Petrohue river (location 9),(iii) fjords, channels and Queulat River (locations 11 to18) and (iv) southern-most fjords and channels (loca-tions 19 to 21). High population structure (FST = 0.85,p < 0.0001) was found among 21 populations and thefour groups defined. No haplotype was shared betweenthe four groups.
We found an overall high haplotype diversity (0.8775+/- 0.0195) but low nucleotide diversity (0.001610 +/-0.000922) for L. provocax. The expected pattern of alower diversity for the glaciated region was notobserved, since the haplotype diversity of the southerngroup (h = 0.7873 +/- 0.0506 Table 2) was high andcomparable to that of the non-glaciated area, such asthe continental freshwater habitat (h = 0.6092 +/-0.0869, Table 2) or the entire northern region (ChiloéIsland + continental rivers and lakes in Chile andArgentina, h = 0.7220 +/- 0.0537).The model selected for the three concatenated genes
was TrN+I, selected based on the lower value of Akaikeinformation criterion (AIC). The Bayesian phylogeneticanalysis (BA; Figure 3) showed two divergent cladesincluding haplotypes described for continental fresh-water habitat, whereas haplotypes from Chiloé Island(A.IV.A) and Nahuel Huapi (A.IV.I) belonged to a poly-tomous group of mixed origins. A continental fresh-water clade is represented by two haplotypes widelydistributed along continental rivers and lakes (A.I.A andA.I.B), and another one by two haplotypes (B.II.A andC.III.A) found on Queule (39°12’S) and Petrohue rivers(41°08’S). Another clade was represented by the majority
Table 1 Sampling sites of Lontra provocax analysed in this study
Localities code Group Locality name Geographical coordinates Sample Size Haplotype
Latitude (S) Longitude (W)
CRL-1 Continental Huilio River, Chile 38°58’ 73°01’ 2 A.I.A
CRL-2 rivers and lakes Queule River, Chile 39°12’ 72°55’ 5 (4) A.I.A; B.II.A
CRL-3 Mahuidanche River, Chile 39°13’ 72°50’ 1 A.I.B
CRL-4 Lingue River, Chile 39°27’ 73°05’ 8 A.I.A
CRL-5 Cua cua River, Chile 39°42’ 71°54’ 3 (1) A.I.A
CRL-6 Riñihue Lake, Chile 39°46’ 72°27’ 2 A.I.A
CRL-7 Traful Lake, Argentina 40°30’ 71°35’ 1 A.IV.I
CRL-8 Nahuel Huapi Lake, Argentina 41°05’ 71°35’ 4 A.IV.I
CRL-9 Petrohue River, Chile 41°08’ 72°24’ 4 C.III.A
CI-10 Chiloé Island Darwin, Chiloé Island 41°52’ 73°39’ 5 (1) A.IV.A
CI-11 Chepu River, Chiloé Island 42°02’ 73°58’ 4 A.IV.A
SFC-12 Southern Fjords, Tictoc Island 43°38’ 73°01’ 2 J.IV.C; D.IV.C
SFC-13 and Channels Melinka 43°53’ 73°44’ (2) A.IV.G
SFC-14 Queulat River 44°27’ 73°35’ 9 E.IV.C; A.IV.C
SFC-15 Seno Magdalena, Magdalena Island 44°35’ 72°56’ 9 (1) A.IV.C
SFC-16 Valle Marta, Magdalena Island 44°52’ 72°55’ 1 A.IV.C
SFC-17 Puyuhuapi Channel, Magdalena Island 44°44’ 72°50’ 4 E.IV.C
SFC-18 Puerto Aguirre 45°09’ 73°31’ (4) F.IV.H, A.IV.G
SFC-19 Madre de Dios Island 50°00’ 75°07’ 1 K.V.D
SFC-20 Around Puerto Natales 51°43’ 72°29’ 1 (1) I.V.D
Sampling sites are divided by habitat type (CRL-continental rivers and lakes, CI- Chiloé Island or SFC-Southern Fjords and Channels) and locality numbers (Figure1, 2), including geographic coordinates, sample size (in parenthesis included the number of samples obtained from tissue), and concatenated haplotypes foundfor each location.
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of haplotypes from the SFC. One of the SFC haplotypes(A.IV.G) was found within the paraphyletic assemblageconsisting of freshwater haplotypes.The divergence between L. felina and L. provocax was
1.5%, whereas it reached 5.2% between L. felina andL. longicaudis, and 4.6% between L. longicaudis andL. provocax. The mean distance within the L. provocaxclade was 0.3%. L. provocax mean distance betweenclades varied from 0.5% between a freshwater clade(B.II.A+C.III.A) and the SFC clade, 0.4% among
continental freshwater clades (A.I.A.+A.I.B and B.II.A.+CI.II.A), to 0.1% among the freshwater lineages (A.I.A.+A.I.B, A.IV.A, A.IV.I, A.IV.G).A signature of recent expansion was detected from the
SFC clade, as evidenced by the significant Fu’s test (Fs=-3.57, P = 0.03), although Tajima was not significant(D= -1.12, P = 0.13). The observed mismatch distribu-tion was not significantly different from that expectedfor both models, however the spatial expansion modelfit the SFC-clade data better than demographic
Table 2 Genetic diversity of mtDNA sequences for Lontra provocax
Geographic areas RC ND5 Cyt-b RC+ND5+Cyt-b - Genetic Diversity
Sample size, polymorphic sites (S), number of haplotypes (Hap) for each of mtDNA sequences (CR, ND5 and Cyt-b), haplotype diversity (h) and nucleotidediversity (π) for the concatenated sequences CR+ND5+Cyt-b by habitat.
1.00
1.00
1.00
1.00
1.00
A.I.A
0.97B.II.A
C.III.A
A.I.B
A.IV.A
A.IV.G
G.VI.E
0.80
0.99
0.84
0.85
0.99
D.IV.CJ.IV.C
A.IV.CE.IV.C
F.IV.HH.IV.F
D.V.D
I.V.D1.00
Lf-A.I.ALf-B.I.B
Lf-T.VI.HLf-U.VI.IL. longicaudis
L. provocax
L. felina
Continental Freshwater habitatChiloé Island Freshwater habitatSouthern Fjords and Channels
0.98K.V.D
A.IV.I
0.005 substitution/site
Figure 3 Bayesian phylogenetic tree constructed using 2151 bp of mitochondrial DNA CR, ND5 and Cyt-b concatenated haplotypes.Four L. felina haplotyes were incorporated on the phylogeny, as well L. longicaudis as outgroup. Nodes support values are presented as Bayesianposterior probabilities. Black colours represent haplotypes from continental freshwater habitat, diagonal black lines represent rivers from ChiloéIsland and gray colour is haplotypes from Southern Fjords and Channels.
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expansion model (SSD P-value = 0.80, SSD = 0.02 forspatial expansion vs. SSD P-value < 0.001, SSD = 0.30for demographic expansion). In contrast, the MedianJoining Network (MJN; Figure 4) topology for the entiredistribution of the SFC haplotypes, which differs fromthat expected in a recent population expansion (i.e. star-like phylogeny with retention of the ancestral haplo-type). MJN indicates high divergence among haplotypesfrom freshwater habitat and a lack of intermediate hap-lotypes. Moreover, the Bayesian skyline plot did notshow any evidence of historical population growth forthe populations from the SFC clade, but rather a recentpopulation decrease (Figure 5). No signature of recentexpansion was detected for L. provocax from the distri-bution in non-glaciated freshwater regions (D= -0.29,P = 0.43; Fs = 1.34, P = 0.78).
DiscussionOur data evidenced a strong genetic differentiationbetween continental freshwater and SFC regions. Thelimits of the latter correspond not only to a habitatchange, but also to a major biogeographic break formarine [14] and freshwater [15] species, and to thenorthern limit of coastal LGM ice cover [2,3].
Southern marine fjords and channels: glacial survival?Population displacements followed by founder effectsdue to recolonization of southern areas from a northern
refuge would produce a signature of reduced geneticdiversity, when compared to northern areas. The latterpattern was described for southern bull kelp (Durvillaeaantarctica), which has reduced genetic diversity insouthern Chile with a clear signature of postglacialexpansion [38]. In our case, phylogenetic reconstructionfor L. provocax indicates that most haplotypes from SFCform a distinctive haplogroup emerging from a basal
B.II.A
C.III.AA.IV.A
A.I.B
A.I.A
I.V.D
D.V.D
G.VI.E
J.IV.C
D.IV.C
E.IV.C
H.IV.F
A.IV.CF.IV.H
A.IV.G
Chiloé Island and Continental Freshwater habitatSouthern Fjords and Channels
K.V.D
A.IV.I
Figure 4 Median Joining Network of CR+ND5+Cyt-b haplotypes. Coloured circles represent haplotypes such as the figure 1 and 2.Haplotypes from the different environments Southern Fjords and Channels, Chiloé Island and Continental Freshwater habitat are indicated.
Figure 5 Bayesian Skyline plot of populations from areascovered by ice sheet during LGM (clade from SFC) showingthe effective population size stability throughout time. X axis:time in years BP, Y axis: is Ne (female effective population size). Themiddle line is the median estimate, and the grey area shows the95% highest probability density (HPD).
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northern haplotype found in freshwater habitat, whichcould suggest a postglacial recolonization from thenorthern freshwater refuge. However, several resultssuggest a different scenario: a non-starlike network ofhaplotypes from SFC, an absence of historical popula-tion growth signature from the skyline plot analysis,high haplotype diversity, and highly divergent lineages.These results are not in agreement with a scenario ofpost-glacial expansion from northern freshwater popula-tions. They rather support the hypothesis of a persis-tence of the species in this region during LGM. Thehypothesis of persistence in glaciated areas is most oftenrejected by phylogeographic studies. Some studies have,however, shown phylogeographic results strongly sup-porting demographic persistence in areas supposedlycovered by ice [39].In Patagonia, iso-pollen lines indicate the existence of
a terrestrial southern refuge [4] along the south-easterncoast of the Island of Tierra del Fuego, east from theBeagle Channel. The presence of a southern refugewould explain the present day plant species disjunctionbetween Chiloé and Magallanes, and the presence ofBryophyta species and vascular herbaceous plants notdistributed further north of 47-48°S [40]. Nevertheless,the unique southern glacial refugium described for ter-restrial species was not supported by our data. Single ormultiple southern refugia and the persistence of the spe-cies within glaciated areas in Chilean Patagonia weredebated for terrestrial [7-9,34,35,41-46] and freshwaterspecies [10-13,47,49]. In the case of temperate forestspecies, patches persisted either at the northern limits ofice cover in Chile or on the eastern limits of ice sheet inArgentinean Patagonia [34,35,42,44,48]. All these exam-ples point to a post-glacial colonization of western Pata-gonia, i.e. the Chilean side of the Andes. The survival ofL. provocax in non-glaciated areas of the eastern side ofthe Andes would have required a recolonization of thespecies across the Andes at multiple sites in order toallow the re-introduction of such a high genetic diver-sity. In addition, because the species currently occursonly in the Nahuel Huapi and Limay River area in conti-nental Argentina [24-26], subsequent localised extinc-tions of L. provocax along most of the eastern refugiawould be required to support such a scenario. Lastly,the unique Argentinean haplotype (A.IV.I) is derivedfrom haplotype A.IV.A present in Chiloe Island, suggest-ing that its distribution was likely widespread in therecent past, and that range expansion was more likelyfrom Chile to the eastern side of the Andes. Thus, theresults do not support an origin of L. provocax diversityfrom eastern Patagonia.On the other hand, glacial refugia on western coast of
the Andes were suggested for some freshwater species.MtDNA sequences of the fishes Galaxias platei and
G. maculatus along the western side of the Andesmountain (39°-49°S) suggest that these species survivedin small southern sites due to discontinuities of the icefield [12,49]. Also, exposed portions of the Pacific conti-nental shelf could have constituted favourable environ-ment for such aquatic species [12]. Similarly, thefreshwater crab Aegla alacalufi seem to have survived inglaciated areas, at least in a site identified as the ElAmarillo hot springs, which was possibly left uncoveredby glacial ice [13].Whether multiple refugia existed or L. provocax sur-
vived all along the western southern Patagonia duringLGM is still a matter of debate. The persistence ofL. provocax is dependent on terrestrial habitat for dens(shelter) and on aquatic habitat for food availability.Such habitat was probably available on the eastern sideof the Andes, as suggested by the extension of the distri-bution of Fitzroya cupressoides [34], allowing the survi-val of L. provocax in a large area and therefore allowingthe persistence of high genetic diversity. In the SFC,L. provocax is known to feed not only on crustaceansand sea urchins, but also on intertidal and subtidalfishes (46% of its diet) [28]. Ice scour can eliminateintertidal and shallow water benthos in the SouthernOcean [50]. In the case of complete elimination of inter-tidal resources, diet could have been based on speciessuch as the freshwater fish (Galaxias platei andG. maculatus), catfish (Trichomycterus areolatus) or thefreshwater crab (Aegla alacalufi), which survived in thearea during LGM [12,13,47,49], or subtidal organisms.Otter species, such as North American river otter (Lon-tra candensis), the Eurasian otter (Lutra lutra) and thesea otter (Enhydra lutris), are distributed throughoutextreme cold environments. L. canadensis inhabiting themarine environments in Alaska has access to two majortypes of prey: intertidal-demersal organisms such asfishes (Cottidae, Hexagrammidae) and crustaceans, andseasonally available schooling pelagic fishes [51]. Simi-larly, L. provocax populations could have shifted theirdiet according to prey availability and thus persistedduring the LGM in the Patagonian SFC. Although spe-cies such as the Eurasian otter (Lutra lutra) show evi-dence of a unique glacial refuge and low geneticdiversity [52], other mustelids were able to survive dur-ing LGM. Gulo gulo, Mustela nivalis and Mustela ermi-nea show adaptations for survival in Pleistoceneconditions [53].
Southern glacial refugia or adaptation to marine habitatOur data show a monophyletic haplogroup for mosthaplotypes from the SFC range of L. provocax (43°S to53°S), distinct from freshwater habitat haplotypes. Suchdifferences between the L. provocax populations inhabit-ing the freshwater and marine environments suggest
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either past genetic isolation, and/or restricted gene flowbetween them at the present time, allowing genetic driftor natural selection to operate. Changes in the L. provo-cax diet and a greater ability to swim larger distances asrequired in the SFC could eventually lead to adaptationto the marine environment, if plasticity is limited. How-ever, patterns of genetic diversity generated by genesurfing during recolonization are similar to those gener-ated by selection and could thus be mistakenly inter-preted as adaptive events [54]. Similarly to selection andunlike most other demographic effects, gene surfinggenerally does not affect all loci, and thus seems espe-cially difficult to distinguish from directional selection[55]. All otter species, except the sea otter (Enhydralutris) and the chungungo (Lontra felina), are dependentof freshwater habitat [20]. Nevertheless, freshwatersources are abundant in SFC and L. provocax can tem-porarily return to rivers to access a supply of freshwater. Moreover, all but four freshwater otter specieshave also been recorded along the coast [20] (Lontracanadensis, Lutra lutra and Lontra longicaudis, amongothers). Nevertheless, no genetic surveys have been con-ducted to determine divergence of otter lineages fromdifferent environments. Lontra phylogeny based onmtDNA markers revealed the recent divergence betweenL. provocax and L. felina about 883,000 years ago (95%HPD: 0.16-1.89 mya) with a possible speciation of L.felina from L. provocax living on SFC [37]. This specia-tion scenario is in agreement with the adaptationhypothesis of L. provocax to the marine habitat in SFC.
Conservation implicationsAlthough higher haplotype diversity was expected alongnorthern populations due to the persistence of riversand forests, low haplotype diversity (compared to SFC)but high divergence among haplotypes was observed.Our results are concordant with the hypothesis of arecent loss of genetic diversity in freshwater environ-ments due to hunting and habitat destruction. This isspecifically supported by: i) A.I.A haplotype sharedamong several locations; ii) two highly divergent clades;iii) two divergent haplotypes (A.I.A and B.II.A) in theQueule River. Genetic theory predicts that during popu-lation bottlenecks low frequency alleles are lost bygenetic drift [56]. Similarly, L. provocax intermediatehaplotypes are not seen on the MJN, suggesting thatthey were eliminated within the freshwater range. Thispattern is consistent with the history of the L. provocaxpopulations in the region. Indeed, L. provocax popula-tions have been eliminated from the north of its pastrange. The northern limit of distribution changed fromCauquenes and Cachapoal rivers (34°S) to Tolten Riverbasin (39°S) [21]. Its small geographical range has beenstrongly impacted by anthropogenic activities resulting
in a decline to less than 10% of its former distributionin freshwater habitats. L. provocax was intensivelyhunted for its fur; and hunting continued until the1970’s in some southern localities [21]. Furthermore, thespecies activity has been significantly reduced in areaswhere riparian vegetation was removed or watercourseswere disturbed or recently polluted by pulp factories[57,58]. Riparian vegetation significantly influences thepresence of crustaceans and consequently the occur-rence of L. provocax in the area [30,32,57].The reduction in the distribution of L. provocax led to
the classification of the species in Chile as ‘’endangered’’in the northern range between the O’Higgins and LosLagos regions, in rivers and lakes, and ‘’insufficientlyknown’’ for the Aysén and Magallanes regions, where dis-tribution was largely marine [59]. Although L. provocaxin freshwater habitats mostly occur below 300 m altitude[33], the majority of National Parks and Reserves (> 90%)in south-central Chile (35.6° to 41.3°S) are located above600 m altitude [48], and therefore do not serve theconservation of this species.Our data shows that the survival of the species along
SFC during glacial cycles maintained a high diversityalong SFC. Large-scale temperate deforestation in Chilehas progressed from north to south [60]. Human popu-lations are concentrated in the central-south region ofChile, and are less dense south of Chiloé Island. Thus,southern L. provocax populations have not been greatlyimpacted by anthropogenic actions, and have main-tained high genetic diversity compared to northernfreshwater populations. L. provocax populations alongSFC are, however, barely studied, and increasing humanactivities in this area are a potential threat to thesepopulations.
ConclusionsOur results evidenced the persistence of a semi-aquaticcarnivore species, the huillín, in western Patagonia alongareas covered by ice sheet during LGM. Marine habitatof the SFC played an important role for L. provocax sur-vival during LGM, probably associated to the survival ofother freshwater and marine species that may haverepresented a persistent food source for the huillín.Therefore, genetic differentiation between northernexclusively freshwater habitat dominated by riparianvegetation and SFC may be explained by some ecologi-cal differentiation between both kinds of habitats. Thisis an interesting clue for understanding why so manyaquatic species seem to have persisted in glaciated areas.
MethodsStudy area, sample collection and DNA extractionA total of 57 feces and 18 tissue samples were collected.These included blood from captured animals, muscle
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from carcasses of animals that died of natural causes,and pelts confiscated by authorities due to illegal hunt-ing. Feces and muscle tissue were preserved in pureethanol. The sampling range included the full range ofthe species (Figure 1) from the northern limit in Chile,the Huilio River tributary of Tolten river (38°58’S) tosouthern Patagonia (53°08’S, Table 1, Figure 1, 2). Inaddition we sample the only known population of theeastern side of the Andes: the Nahuel Huapi Area.A total of 21 locations were sampled, nine in continen-tal rivers and lakes (CRL), two in rivers from ChiloéIsland (CI) and ten marine locations in the fjords andchannels (SFC).
PCR amplificationMitochondrial DNA control region (CR), NADH dehy-drogenase subunit 5 (ND5) gen and cythocrome b (Cyt-b) gen were amplified using primers described by [37]:LfCR-2F and LfCR-1R; ND5-DF1 and LfND5-R; andLfCYTB-1F; LfCYTB-1F; LfCYTB-2F and LfCYTB-2R.PCR reactions were carried out following [37]. PCR
amplification was confirmed by electrophoresis of pro-ducts with ethidium bromide in 0.8% agarose gels andvisualization under UV light. Amplicons were purifiedusing QIAquick PCR purification kit (Qiagen) andsequenced using amplification primers by MacrogenInc., Seoul, Korea. All sequences have been deposited inthe GenBank [GenBank: GQ843803-GQ843824, andHM997011-HM997017].
Data analysisPopulation analysisThe sequences were aligned and mutations were con-firmed by eye according to the chromatogram usingProseq ver. 2.91 [61]. All sequences were aligned andhaplotypes were identified using ClustalX ver. 1.83 [62].Spatial analysis of molecular variance (SAMOVA) was
implemented by SAMOVA ver. 1.0 [63] to define groupsbased on the geographic distribution of the geneticdiversity. SAMOVA were performed for 21 locationstesting from 2 to 7 groups, each of which with 100initial conditions. The groups of populations geographi-cally homogeneous are defined by maximizing FCT
values (among group variance) and minimizing FSC
values (among populations within group variance). Hap-lotype (h) and nucleotide diversity (π) were calculatedusing Arlequin program ver. 3.0 [64] for all data set andfor the different environments.Deviations from a neutral Wright-Fisher model were
performed by calculating Tajima’s D and Fu’s Fs statis-tics [65,66]. We tested the demographic [67] and spatialexpansion [68] models by calculating the sum ofsquared differences (SSD) between the observed and anestimated mismatch distribution obtained by 1,000
bootstrap. The P-value of the SSD statistic was calcu-lated as the proportion of simulated cases that show aSSD value distinctive from the original. Calculationswere performed in ARLEQUIN, using 1,000 bootstrapto evaluate significance. To estimate the shape of popu-lation growth through time for the individuals distribu-ted along the ice sheet coverage area during the LGM,we constructed Bayesian skyline plots implemented inBEAST v 1.4.8 [69]. The appropriate model of nucleo-tide substitution was HKY+I determined using ModelT-est ver. 3.06 [70]. Five million iterations wereperformed, of which the model parameters weresampled every 1000 iterations. Throughout our analysis,we assumed a within-lineage per site mutation rate of6%Ma. Demographic plots for each analysis were visua-lized using Tracer v1.0.1 [69].Phylogenetic analysisWe applied the Partition Homogeineity test (10,000 per-mutation) to assess the congruence of the evolutionrates among CR, ND5 and Cytb using PAUP ver. 4.0b8[71]. The evolutionary relationship among concatenatedCR+ND5+Cyt-b haplotypes was investigated by a Med-ian Joining Network using Network ver. 4.5.1.0 [72], andphylogenetic reconstructions based on Bayesian (BA)methods. Four divergent haplotypes of Lontra felina[37], were incorporated in the phylogenetic reconstruc-tion, whereas a L. longicaudis haplotype from the Ama-zon (this manuscript) was used as an outgroup. Thesubstitution model of DNA evolution was selected basedon AIC using Modeltest ver. 3.06 [69]. BA was per-formed by MrBayes ver. 3.1.2 [73] using the generaltype of the best fit model parameters defined for thedata set, in which four independent analyses were runwith four chains each, for six million generations andthen sampled at intervals of 1,000 generations. The first25% of sampled trees were discarded to ensure stabiliza-tion and the remaining used to compute a consensustree. The split frequency was below 0.004, confirmingthat sampling was from the posterior probability distri-bution. Mean distance between clades and species wascalculated using Mega v.3.1 [74] using p-distance.
AcknowledgementsThis work was supported by Universidad Andrés Bello-DI-06-06/R, RuffordSmall Grant for Nature Conservation, Earthwatch Institute and FONDECYT1100139. Vianna was supported by a CONICYT Doctoral Fellowship, CONICYTThesis Project AT-23070034. Special thanks to René Monsalves, Attia Zerega,Juan Carlos Marín, Gerardo Porro, Carla Pozzi, Javier Lucotti who helped withsample collection. Samples from southern Patagonia were collected byServicio Agricola y Ganadero (SAG) after illegal hunting. Florance Tellier,Andrés Parada and Emma Newcombe helped with analysis and English. AllChilean samples were collected according to permits: Subsecretaria de Pesca(686-2006; 1588-2009; 1228-2009).
Author details1Departamento de Ecología y Biodiversidad, Facultad de Ecología y RecursosNaturales, Universidad Andrés Bello, Republica 440, Santiago, Chile. 2Center
Vianna et al. BMC Evolutionary Biology 2011, 11:53http://www.biomedcentral.com/1471-2148/11/53
for Advanced Studies in Ecology and Biodiversity, Facultad de CienciasBiológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago,Chile. 3Departamento de Ecosistemas y Medio Ambiente, Facultad deAgronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile,Vicuña MacKenna 4860, Santiago, Chile. 4Delegación Regional Patagonia,Administración de Parques Nacionales, Vice Almirante O’Connor 1188, 8400,San Carlos de Bariloche, Argentina. 5Universidad Arturo Prat, Departamentode Ciencias del Mar, Av. Arturo Prat 2120, Iquique, Chile. 6Fundación CEQUA,Plaza Muñoz Gamero 1055, Punta Arenas, Chile.
Authors’ contributionsJAV conceived of the study, participated in sample collection, moleculargenetic studies and in statistical analyses and drafted the manuscript. GMVparticipated in the design of the study and helped to draft the manuscript.CC helped on the field work and results interpretation based on hisexperience on the species to draft the manuscript. WS helped on the fieldwork and results interpretation based on his experience on the species todraft the manuscript. CO helped on the field work and to draft themanuscript. SF participated in the design of the molecular genetic studies,contributed to the discussion of results and to the interpretation anddrafted the manuscript. All authors read and approved the final manuscript.
Received: 1 September 2010 Accepted: 28 February 2011Published: 28 February 2011
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doi:10.1186/1471-2148-11-53Cite this article as: Vianna et al.: Phylogeography of the Patagonianotter Lontra provocax: adaptive divergence to marine habitat orsignature of southern glacial refugia? BMC Evolutionary Biology 201111:53.
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